Method for the identification of drug targets

ABSTRACT

The present invention relates to the field of drug development. More specifically the invention provides a method for the identification of drug targets. The method can also be used for analysis of proteomes. The method utilizes in essence a combination of two chromatographic separations of the same type, separated by a step in which the population of the drug-bound targets is altered specifically on the drug in such a way that the chromatographic behaviour of the altered drug-bound targets in the second chromatographic separation differs from the chromatographic behaviour of its unaltered version. The different chromatographic behaviour of the altered drug-bound targets is used for the isolation and subsequent identification of the targets.

FIELD OF THE INVENTION

The present invention relates to the field of drug development. Morespecifically the invention provides a method for the identification ofdrug targets. The method can also be used for analysis of proteomes. Themethod utilizes in essence a combination of two chromatographicseparations of the same type, separated by a step in which thepopulation of the drug-bound targets is altered specifically on the drugin such a way that the chromatographic behaviour of the altereddrug-bound targets in the second chromatographic separation differs fromthe chromatographic behaviour of its unaltered version. The differentchromatographic behaviour of the altered drug-bound targets is used forthe isolation and subsequent identification of the targets.

BACKGROUND OF THE INVENTION

Now in the post-genome era, many strategies for the analysis of proteinsare currently being developed. Most conventional approaches focus onrecording variations in protein level. These approaches are commonlyreferred to as “proteomics”. In general, proteomics seeks to measure theabundance of broad profiles of proteins from complex biologicalmixtures. In the most common embodiments, proteomics involves separatingthe proteins within a sample by two-dimensional SDS-PAGE. Then, theindividual protein spot patterns of these gels can be compared to getindications as to the relative abundance of a particular protein in twocomparative samples. The approach can even be extended to determine themolecular identity of the individual protein spots by excising the spotsand subjecting them to peptide mass fingerprinting. More recently,methods have been described for eliminating the electrophoresis stepsand performing proteomics by directly analyzing the complex mixture bymass spectrometry. For example, methods currently described in the artprovide chemically reactive compounds that can be reacted with a proteinmixture to label many proteins in that mixture in a non-specific, ornon-directed, manner providing only a quantitative analysis of proteins(Link et al. (1999) Nat. Biotechnol. 17, 676-682, Gygi et al. (1999)Nat. Biotechnol. 17, 994-999). Such methods teach that there are manychemically reactive amino acid residues within a protein which can beconjugated to chemical probes, whereby the resulting protein complexescan be subsequently quantified to yield an indication of proteinabundance. In WO 01/77668 the use of activity-based probes (ABP) isdescribed to screen for target proteins of said ABPs. In this technologythe ABPs are coupled with an affinity ligand that serves to detect thedrug-target complexes. There is however an urgent need to developmethods that allow a more detailed analysis of a complex mixture ofproteins or even of a whole proteome. It is well known that the control(activation or inhibition) of protein activities in a cell is due tochanges in the protein structure available to other components in thecell. Conformational changes and movements in hinge regions of proteinsexpose specific parts of these proteins and allow them to contactcompounds such as enzyme substrates, adaptor proteins, and othercomponents such as drugs. Moreover, the activity of drugs is due to thespecific interaction with proteins influencing their biologicalactivity. In several cases the protein targets of existing drugs areknown: e.g. aspirin reacts with the cyclo oxygenases, penicillin is apseudo substrate of the peptide glycan amino transferase ofGram+bacteria, etc. Also in some exceptional cases, drugs have beendesigned and improved based on the 3D-structure of the target protein.In most cases, however components with biological activities have notyet been allocated to their target proteins and hence the targets ofmost drugs are unknown. The reliable identification of the targets ofexisting drugs or drugs in development would be extremely valuable forthe estimation of the specificity and prediction of side effects ofdrugs. Furthermore it is known that the inter-individual response todrugs varies considerably. The aim of modern drug development is togenerate tailor made drugs that are efficient for individual patientcategories. The present invention relates to a solution to theabove-cited problems and discloses a method to determine the interactionpartners of drugs and also the interaction site in the primary structureof the target protein. The method can be used to estimate a correlationbetween the disease response to a certain drug with the targets of saiddrug identified in individual patients or patient groups. Our method isindependent of the use of detectable or affinity labels that are coupledto the drugs, as described in WO 01/77668. In addition, the methodoffers the advantage that the drug targets can be efficiently isolatedin a chromatographic step. In addition the site in the primary structureor the protein target on which the drug binds can be efficientlydetermined with the current invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A): Actin was incubated with a target peptide “CP” andcross-linked by transglutaminase. The cross-linked components weredigested with endo-Lys-C. The UV-absorption profile of endo-Lys-Cpeptides separated on a C-18 reversed phase column (run 1) is shown inFIG. 1A. Solvent A is 0.1% TFA, solvent B is 70% acetonitrile in 0.1%TFA-water. The gradient of solvent B is indicated. Eluting peptides arecollected in 5 min. wide intervals and dried B). Fraction 6, containingthe cross-linked peptide was rerun in the same chromatographicconditions as in run 1 after specific cleavage with factor Xa. Theshifted peptide carrying the cross-link is visible in FIG. 1B in frontof the bulk of unmodified peptides (in black).

FIG. 2: The cross-linked peptide, shifting in front of fraction 6 (FIG.1B) was analysed by electrospray ionization mass spectrometry. Thedifferently charged peptide ions are shown and allow the determinationof the mass of this cross-linked dipeptide. The analysis was carried outon a Micromass Q-TOF apparatus.

FIG. 3A): A total lysate of Jurkat cells was digested with endo-Lys-C.This peptide mixture was mixed with a similar digest of the actin-CPconjugate. The peptide mixture was separated by reversed-phasechromatography as in FIG. 1A. The first part of the chromatogram wasrecorded at AUFS 0.1, the second part at AUFS 0.2. The eluate wascollected in fractions of 2 min. These fractions were dried andrecombined as in Table 1 before being treated with factor Xa.

FIG. 3B) shows the UV traces of the peptides in pool D (see Table 1).The profiles of primary fractions 9, 14 and 19 are shown. 9* is a peakeluting in front of the bulk of peptides. 9** is peptide Ac-F-1-E-G-Rderived from excess of CP and cleaved by factor Xa. Note a peak (indark) eluting in front of fraction 14. All chromatographic conditionswere as in the experiment of FIG. 1.

AIMS AND DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an alternative method for the isolationand identification of drug targets. The method also allows thequantitation of expression levels and/or activities of classes ofproteins or/and enzymes or individual proteins or/and enzymes in aglobal cell lysate background. The method utilizes in essence acombination of two chromatographic separations of the same type,separated by a step in which the population of the drug-bound targets isaltered specifically on the drug in such a way that the chromatographicbehaviour of the altered drug-bound targets in the secondchromatographic separation differs from the chromatographic behaviour ofits unaltered version. The different chromatographic behaviour of thealtered drug-bound targets is used for the isolation and subsequentidentification of the targets.

In one embodiment the invention provides a method to isolate at leastone target molecule of a compound comprising a functional group that canbe specifically altered, said method comprises the following steps (a)adding said compound to a complex mixture of molecules wherein saidcompound stably interacts with at least one molecule forming acompound-target complex, (b) separating the resulting complex mixture ofmolecules and compound-target complexes into fractions viachromatography, (c) chemically, or enzymatically, or chemically andenzymatically altering said compound present on at least onecompound-target complex in each fraction, and (d) isolating at least onetarget molecule that interacts with said compound via chromatography,wherein the chromatography of steps (b) and (d) is performed with thesame type of chromatography.

In another embodiment the invention provides a method to isolate atleast one target protein of a compound comprising a functional groupthat can be specifically altered. Said method comprises the followingsteps (a) adding said compound to a complex mixture of proteins whereinsaid compound stably interacts with at least one target protein forminga compound-protein complex, (b) separating the resulting complex mixtureof proteins and compound-protein complexes into fractions viachromatography, (c) chemically, or enzymatically, or chemically andenzymatically altering said compound present on at least onecompound-protein complex in each fraction, and (d) isolating at leastone target protein that interacts with said molecule via chromatography,wherein the chromatography of steps (b) and (d) is performed with thesame type of chromatography.

In another embodiment the invention provides a method to isolate atleast one target peptide of a compound comprising a functional groupthat can be specifically altered. Said method comprises the followingsteps (a) adding said compound to a complex mixture of proteins whereinsaid compound stably interacts with at least one target protein forminga compound-protein complex, (b) cleaving the resulting complex proteinmixture and compound-protein complexes into a protein peptide mixture,(c) separating said protein peptide mixture into fractions viachromatography, (d) chemically, or enzymatically, or chemically andenzymatically altering said compound present on at least onecompound-peptide complex in each fraction and (e) isolating at least onetarget peptide that interacts with said compound via chromatographywherein the chromatography of steps (c) and (e) is performed with thesame type of chromatography.

In yet another embodiment the invention provides a method to isolate atleast one target of a compound comprising a functional group that can bespecifically altered wherein said compound is added directly to aprotein peptide mixture and wherein said compound stably interacts withat least one target peptide forming a compound-peptide complex.

In yet another embodiment the chromatographic conditions used in thepreceding methods are the same or substantially similar.

As used herein, a “protein peptide mixture” is typically a complexmixture of peptides obtained as a result of the cleavage of a samplecomprising proteins. Such sample is typically any complex mixture ofproteins such as, without limitation, a prokaryotic or eukaryotic celllysate or any complex mixture of proteins isolated from a cell or aspecific organelle fraction, a biopsy, laser-capture dissected cells orany large protein complex such as ribosomes, viruses and the like. Itcan be expected that when such protein samples are cleaved into peptidesthat they may contain easily up to 1.000, 5.000, 10.000, 20.000, 30.000,100.000 or more different peptides. However, in a particular case a“protein peptide mixture” can also originate directly from a body fluidor more generally any solution of biological origin. It is well knownthat, for example, urine contains, besides proteins, a very complexpeptide mixture resulting from proteolytic degradation of proteins inthe body of which the peptides are eliminated via the kidneys. Yetanother illustration of a protein peptide mixture is the mixture ofpeptides present in the cerebrospinal fluid.

The term ‘at least one target of a compound’ means that a particularcompound stably interacts with one or more target molecules, or a classof molecules. The binding of a compound to the target is specific,meaning that said compound binds to at least one molecule in a complexmixture of molecules and not to other molecules. Usually a compound is adrug, a drug analogue or drug derivative. Preferably said binding causesan inactivation or a partial inactivation of the molecule (e.g. inhibitsits activity) and the binding preferably occurs at the active site ofthe molecule (e.g. of a protein). Since the binding occurs at the activesite of a protein the method of the present invention can also be usedfor the isolation of a specific class of active proteins. Active meansthat the active site is accessible for the compound whereas inactiveproteins of the same class will not be isolated because the active siteis not accessible for the compound.

Here an ‘active site’ of a protein refers to the specific area on thesurface of a protein (e.g. an enzyme or receptor), to which a compound(e.g. a substrate, a ligand, a drug or a drug analogue or a drugderivative) can bind resulting in a change in the configuration of theprotein. With regard to a receptor, due to the conformational change,the protein may become susceptible to phosphorylation ordephosphorylation or other processing. With regard to other proteins theactive site will be the site(s) where the substrate and/or cofactor ordrug or drug analogue or drug derivative binds or where the substrateand cofactor undergo a catalytic reaction, or where two proteins form acomplex, (e.g. two kringle structures bind, sites at which transcriptionfactors bind to other proteins, sites at which proteins bind to specificnucleic acid sequences, etc.).

The ‘compounds’ of the invention are chemical reagents that arepoly-functional agents for non-competitive or substantially irreversiblebinding to a target molecule. ‘Compounds’ comprise small compounds(organic or inorganic), existing drugs, drugs in development, drugleads, drug analogues or drug derivatives. An individual compound, asubset of compounds or the complete set of compounds derived from alibrary of compounds such as a library established by combinatorialchemistry. In most general terms, the compound consists of (1) achemical structure determining the specific interaction between saidcompound and its target molecule (the “S”-part), (2) a chemicallyreactive group by which the compound and its target can be tightlycross-linked (the “L”-part) and (3) a functional group which can bealtered on a specific and controllable manner (the “A”-part). Thesethree properties (“S” for specificity, “L” for cross-linking and “A” foralteration) can be differently distributed over the compound structure.

According to the invention a compound-target complex is chemically, orenzymatically, or chemically and enzymatically altered between the twochromatographic separations. In a preferred embodiment a compound is adrug, a drug analogue or drug derivative. A drug derivative is a drug(for example an existing drug) on which an extra group is attached suchas for example an alteration part (“A” part) or a functional group bywhich the compound and its target can be tightly cross-linked (“L”part). Said “A” group or “A” part is necessary and sufficient for thechemical or enzymatic or chemical and enzymatic alteration between thetwo chromatographic separations.

In order to distinguish the “S”, “L” and “A” part of a target moleculefrom the one-letter notation of the amino acids Ser (S), Leu (L) and Ala(A) used in this description of the invention; S, L and A will be usedto define their corresponding amino acids, while “S”, “L” and “A”, or“S”-part, “L”-part and “A”-part, or “S”-moiety, “L”-moiety and“A”-moiety will be used to indicate functional entities within thecompounds: “S”; determining the specificity of the reaction,“L”-determining the group responsible for creating the covalent or tightlink between compound and target molecule and “A”; determining the groupthat can be specifically altered.

While “S”, “L” and “A” could be different entities within the compound,they could share identical functions, either as couples or all threetogether.

In the following examples, different “SLA” components will beillustrated.

The specificity-determining part (the “S”-part) of the compound consistsof a functional group or an assemblage of functional groups comprising achemical moiety interacting with a particular conformation of the target(e.g. the active site of an enzyme). Due to this interaction, thecomplete compound is brought in close contact with the target allowingthe linking being established at reasonable concentrations of thecompound. It is well known that increasing concentrations of thecompound will decrease the specificity. Thus the “S”-part of thecompound should interact with its target under physiologically relevantconcentrations. In some situations the compound “S”-part will be able todiscriminate the active from the inactive target. Meaning that certaincompounds (e.g. drugs) will only target active forms of proteins or,more rarely, others will only target inactive proteins. In othersituations, the conformation of the target protein(s), whereby with areactive functionality or one that requires activation, the predominantreaction will be at the active site. The compound also contains achemically reactive group (“L”-part) which reacts with a functionalitypresent in the target protein. The link between said compound and itstarget is most ideally of covalent nature. However, any binding which issufficiently strong and resistant against all chemical and/or enzymatictreatments, against solvents and buffers used in all chromatographicsteps and against all other steps used in the entire sorting procedurecould be considered. Such non-covalent, but sufficiently strong bindingcan for example be formed between coplanar cys-hydroxyl groups andboronic acid derivatives. The “L”-part could be embedded in the “S”-partof the compound as for instance for the enzyme suicide inhibitors suchas penicillin, 5-fluorouracil, or the caspase-1 inhibitor. Thespecificity-determining group and the linking group should notnecessarily be present in the same moiety, but could be spatiallyseparated in the compound structure. This is illustrated in example 1.4where the “S”-part and “L”-part contact different surfaces at the targetprotein. Such chemically reactive group can be a photo-activatable groupsuch as a diazoketone, arylazide, arylketone, arylmethylhalide, etc. anyof which can bind non-selectively to a target protein, but which istransferred by the “S”-part at a specific site of the target protein.Such chemically reactive group can consist of a functional group withhigher selectivity. Selectivity for amino-groups such as amidates,succinic acid anhydride and the like; for SH-groups such asmethylmaleimide or acetylhalides and the like. Such chemically reactivegroups may form links which can be broken afterwards. For instance,bonds formed between maleic acid anhydride and amino-groups may bebroken by acid treatment. Such links between the “L”-part and the targetprotein may be formed by enzymatic catalysis. For instance, linksbetween a glutamine side chain on the target and a lysine εNH₂-group onthe compound could be formed by the action of a transglutaminase.

In particular embodiments, the biological target molecule is apolypeptide, a nucleic acid, a carbohydrate, a nucleoprotein, aglycopeptide or a glycolipid, preferably a polypeptide, which may be,for example, an enzyme, a hormone, a transcription factor, a receptor, apeptide ligand for a receptor, a growth factor, an immunoglobulin, asteroid receptor, a nuclear protein, a signal transduction component, anallosteric enzyme regulator, and the like. The biological target canalso be a class or family of polypeptides, nucleic acids, carbohydrates,glycopeptides, or glycolipids, preferably a class of proteins such ashydrolases, dehydrogenases, ligases, transferases and proteins that bindto each other or to other biological structures.

The term “altering” or “altered” or “alteration” as used herein inrelation to a compound-target complex (e.g. a drug-protein interaction),refers to the introduction of a specific modification in the compound(e.g. a drug), with the clear intention to change the chromatographicbehaviour of such a compound-target complex containing said alteredcompound. Usually the alteration is in the “A” part of the compound(alteration part) but the alteration can also take place in the “S or L”part of the compound (specificity or linking part). Such alteration canbe a stable chemical or enzymatical modification. Such alteration canalso introduce a transient interaction with a molecule. Typically analteration will be a covalent reaction, however, an alteration may alsoconsist of a complex formation between the compound bound on the target,provided this complex is sufficiently stable during the chromatographicsteps. Typically, an alteration results in a change in hydrophobicity ornet charge such that the altered compound-target migrates differentlyfrom its unaltered version in revered phase chromatography.Alternatively, an alteration results in a change in the net charge of acompound-target complex, such that the altered compound-target complexmigrates different from its unaltered version in an ion exchangechromatography, such as an anion exchange or a cation exchangechromatography. Alternatively, a specific change in the net charge of acompound-target complex may be equally exploited by electrophoreticsystems, more particularly by capillary electrophoresis. Also thealteration may be the cleavage of a part of the drug-target complex, forexample the “A” part of the drug-target complex. Also, an alteration mayresult in any other biochemical, chemical or biophysical change in acompound-target complex such that the altered compound-target complexmigrates different from its unaltered version in a chromatographicseparation. The term “migrates differently” means that a particularaltered compound-target complex elutes at a different elution time inrun 2 with respect to the elution time of the same non-alteredcompound-target complex in run 1. Such alterations could induce either aforward or backwards shift of the sorted complex in the secondary run.The alteration step should be more specific for the compound-targetcomplex and should not take place on more than one or on more than alimited set of peptides which do not carry the compound. In this casethe altered compound-target complex could be distinguished from thealtered peptides by differential analysis. Preferably, the alterationstep is highly specific for the compound-target complex and does nottake place on any other peptide that does not carry the compound.

Altering can be obtained via a chemical reaction or an enzymaticreaction or a combination of a chemical and an enzymatic reaction of thecompound. A non-limiting list of chemical reactions includes alkylation,acetylation, nitrosylation, oxidation, hydroxylation, methylation,reduction, hydrolysis (basic or acid) and the like. A non-limiting listof enzymatic reactions includes treating the compound-target complexwith phosphatases, acetylases, glycosidases, specific proteinases orother enzymes which modify co- or post-translational modificationspresent on compounds. The chemical alteration can comprise one chemicalreaction, but can also comprise more than one reaction such as forinstance two consecutive reactions in order to increase the alterationefficiency. Similarly, the enzymatic alteration can comprise one or moreenzymatic reactions. Such alteration is applied in between twochromatographic separations of the same type.

The resulting altered product is ideally a peptide carrying an alteredmolecule (a tag) at the site of the original covalent or tight bond.Ideally such a tag should be small and contain a limited number of atomsin order to allow an easy and accurate analysis and identification. Moreideally, although not absolutely necessary, such tag should contain afunctional group which can be labeled either with heavy or light stableisotopes facilitating quantitative differential analysis by massspectrometry.

The term ‘stably interacts’ refers to the interaction between a compound(e.g. a drug or drug derivative) added to a complex mixture of molecules(e.g. a complex protein mixture or a protein peptide mixture). Saidinteraction is strong enough for the isolation of a partner for saidcompound, in other words a target molecule for said compound. Theinteraction is sufficiently stable during the two chromatographicseparations. In a particular embodiment said interaction is a covalentinteraction.

The same type of chromatography means that the type of chromatography isthe same in both the initial separation and the second separation. Thetype of chromatography is for instance in both separations based on thehydrophobicity of the molecules (e.g. peptides) and compound-moleculecomplexes. Similarly, the type of chromatography can be based in bothsteps on the charge of the molecules (e.g. peptides) and the use ofion-exchange chromatography or capillary electrophoresis. In stillanother alternative, the chromatographic separation is in both stepsbased on a size exclusion chromatography or any other type ofchromatography.

The first chromatographic separation, before the alteration, ishereinafter referred to as the “primary run” or the primarychromatographic step” or the “primary chromatographic separation” or“run 1”. The second chromatographic separation of the altered fractionsis hereinafter referred to as the “secondary run” or the “secondarychromatographic step” or the “secondary chromatographic separation” or“run 2”.

In a preferred embodiment of the invention the chromatographicconditions of the primary run and the secondary run are identical or,for a person skilled in the art, substantially similar. Substantiallysimilar means for instance that small changes in flow and/or gradientand/or temperature and/or pressure and/or chromatographic beads and/orsolvent composition is tolerated between run 1 and run 2 as long as thechromatographic conditions lead to the same or predictable elution ofthe unaltered molecules in run 2 and to an elution of the alteredcompound-target complexes (e.g. altered drug-protein or altereddrug-peptide complexes) that is predictably distinct from the unalteredmolecule-target complexes and this for every fraction collected fromrun 1. Altered compound-target complexes have a differentchromatographic behaviour in run 2. The alteration induces a shift ofthe altered compound-target complexes. Due to this shift the alteredcompound-target complexes elute at a different positioning run 2, ascompared to run 1, and consequently said complexes can be isolated andidentified (see further herein).

In a particular example were protein targets of a particular compoundare sought to be determined, after the addition of said compound to aprotein peptide mixture, and separating said treated protein peptidemixture into fractions via a primary chromatographic step, the currentinvention requires that the alteration of compound-peptide complexes iseffective in each of the peptide fractions from the primary run. In afraction derived from said primary run (in a first chromatographic step)peptide and unaltered compound-peptide complexes can be found. Thus, ineach fraction obtained from the primary chromatographic step, thealtered compound-peptide complexes have to migrate distinctly from theunaltered compound-peptide complexes in the secondary chromatographicstep. The alteration of the compound part of the compound-peptidecomplexes induces a shift in the elution of said alteredcompound-peptide complex. Depending on the type of applied alteration,the shift may be caused by a change in the hydrophobicity, the netcharge and/or the affinity for a ligand (e.g. a metal ion) of thealtered compound-peptide complexes. This shift is called δp and isspecific for every individual altered compound-peptide complex. In theexample of a change in hydrophobicity, δp-values can be expressed aschanges in the hydrophobic moment, or as a percentage of organicsolvents in chromatographic runs, but most practically in time unitsunder given chromatographic/electrophoretic conditions. Thus δp is notnecessary identical for every altered compound-peptide complex and liesin-between δ_(max) and δ_(min). δp is affected by a number of factorssuch as the nature of the induced alteration, the nature of the columnstationary phase, the mobile phase (buffers, solvents), temperature andothers. All δp values taken together delineate the extremes of δ_(max)and δ_(min). Given t₁ and t₂, the times delineating the beginning andthe end of the interval of the shifted altered compound-peptidecomplexes, and t₃ and t₄, the times enclosing the fraction taken fromthe primary run, then δ_(min) (the minimal shift) will be determined byt₃−t₂, while δ_(max) (the maximal shift) will be determined by t₄−t₁.Window W₁ is the fraction window in which the unmodified peptides elutein the secondary run w₁=t₄−t₃. Window w₂ is the window in which thealtered compound-peptide complexes will elute w₂=t₂−t₁. Thus:δ_(min)=t₃−t₂; δ_(min)=t₄−t₁; w₁=δ_(max)+t₁−δ_(min)−t₂ andw₂=t₂−t₁=δ_(max)−δ_(min)−w₁. Important elements in the sorting processare: δ_(min), delineating the distance between the unaltered and theleast shifted of the altered compound-peptide complexes in a givenfraction and w₂, the time-window in which altered compound-peptidecomplexes are eluted. The word ‘sorted’ is in this invention equivalentto the word ‘isolated’. δ_(min) has to be sufficient to avoid thataltered compound-peptide complexes elute within window w₁ (and as suchwould overlap with the unaltered compound-peptide complexes), and thisrule should apply for every fraction collected from the primary run.Preferentially δ_(min) should be w₁ or larger in order to minimizeoverlap between altered and unaltered compound-peptide complexes. Forinstance, if w₁=1 minute, min should by preference be 1 minute or more.Avoiding overlap or co-elution of altered compound-peptide complexesimproves the possibility of identifying an optimal number of individualaltered compound-peptide complexes. From this perspective, the size ofwindow w₂ has an impact on the number of altered compound-peptidecomplexes that can be identified. Larger values of w₂ result in adecompression of the altered compound-peptide complex elution time,providing a better isolation of altered compound-peptide complexes and abetter opportunity for analysis by gradually presenting the targets(altered compound-peptide complexes) for identification to analyserssuch as mass spectrometers. While window w₂ may be smaller than w₁, in apreferred embodiment, w₂ will be larger than w₁. For instance if w₁=1minute, w₂ can be 1 minute or more. It is preferred that the size of w₂,and the value of δ_(min) and δ_(max) are identical or very similar forevery fraction collected from the primary run. It is howeverself-evident that minor contaminations of unaltered compound-peptidecomplexes in the elution window of the altered compound-peptidecomplexes is not preferred, but it is acceptable. Manipulation of thevalues of δ_(min), δ_(max) and w₂ to obtain optimal separation of thealtered compound-peptide complexes from the unaltered compound-peptidecomplexes in each primary run fraction is part of the current inventionand comprises, among others, the right combination of the compoundselected for alteration, the type of alteration, and the chromatographicconditions (type of column, buffers, solvent, etc.). While the aspectsof the hydrophilic shift have been worked out herein above, a similardescription could also be provided where a hydrophobic shift was inducedin order to separate the altered compound-peptide complexes from thenon-altered compound-peptide complexes. Here t₃ and t₄ define window w₁in which the unaltered compound-peptide complexes elute, while t₅ and t₆define the window w₂ in which the altered compound-peptide complexeselute. The maximum hydrophobic shift δ_(max)=t₆−t₃, the minimumshift=t₅−t₄. It will be appreciated that similar calculations forconditions in which fractions are pooled may be used.

It is obvious for a person skilled in the art that the same approach canbe applied to isolate compound-peptide complexes with for instance ionexchange chromatography or other types of chromatography.

Thus in case of a complex mixture of peptides (e.g. a protein peptidemixture) in which the compound is only linked to one target peptide orto a limited number of target peptides, while the vast majority ofpeptides is not conjugated to the compound, then the sorting process isas follows. The total peptide mixture is first separated in the primarychromatographic step. The eluting peptides are collected in anappropriate number of fractions. Then, the alteration step is carriedout, for example on the ‘A’-part of the compound-peptide complexespresent in each collected fraction. In principle every fraction issubjected to a second chromatographic step. Peptides linked to thecompound (so called compound-peptide mixtures) will be altered and showa chromatographic shift. Peptides not linked to the compound will elutein the same predictable position during run 2 with respect to run 1.Since every fraction of run 1 occupies only a fragment of the totalseparation protocol of run 2, we can combine multiple fractions of run1, for sorting in run 2. The fractions are combined in such a way, thatthe sorted peptides (here the compound-peptide complexes) do not overlapwith the non-altered peptides of neighbouring fractions. Thus in yetanother embodiment, the invention is directed to the use of a sortingdevice that is able to carry out the method of the invention. As anon-limited example were the molecules of the invention are proteins orpeptides, the method may comprise two consecutive chromatographic steps:a primary chromatographic step using for example a protein peptidemixture (to which a compound, comprising a functional group that can bealtered, with a specificity for a particular peptide or class ofpeptides has been added) which divides said mixture into fractions, anda second chromatographic step that is performed after the specificchemical and/or enzymatic alteration of at least one compound-peptidecomplex present in the fractions. As described herein, the term “peptidesorter” refers to a device that efficiently separates the alteredcompound-peptide complexes from the non-altered complexes. In apreferred aspect, identical or very similar chromatographic conditionsare used in the two chromatographic steps such that during the secondrun the non-altered compound-peptide complexes stay at their originalelution times and the altered compound-peptide complexes are induced toundergo a shift in the elution time. Additionally in another preferredaspect we assume that the alteration of compound-peptide mixtures occursclose to completeness. As described herein, the use of for example apeptide sorter particularly refers to the pooling of fractions obtainedafter run 1 and the optimal organisation of the second chromatographicstep (e.g., the step in which the altered compound-peptides complexesare separated from the non-altered complexes to speed up the isolationof the altered compound-peptide complexes out of each of the run 1fractions). One approach to isolate and identify alteredcompound-peptide complexes isolated from a protein peptide mixture, isto independently collect every fraction from the primary chromatographicseparation, to carry out the chemical and/or enzymatic alteration on thecompound-peptide complexes in each of the fractions and to rerun everyfraction independently in the same chromatographic conditions and on thesame or substantially similar column. Subsequently the alteredcompound-peptide complexes of each independently run secondary run arecollected and passed to an analytical instrument such as a massspectrometer. However, such approach requires a considerable amount ofchromatography time and occupies important machine time on the massspectrometer. In order to obtain a more efficient and economic use ofboth the chromatographic equipment and the mass spectrometer, thepresent invention provides the use of peptide sorters allowing thepooling of several fractions of the primary chromatographic separationwhile avoiding elution overlap between altered compound-peptidecomplexes originating from different fractions, and between alteredcompound-peptide complexes from one fraction and peptides from one ormore other fractions. In each fraction obtained from the primarychromatographic step, altered compound-peptide complexes elute distinctfrom the unaltered complexes. When several fractions of the primary runare combined (pooled), then it is important that during the second runwith the pooled fractions, the sorted altered compound-peptide complexesfrom one selected fraction do not co-elute with the (unaltered) peptidesof one of the previous fractions. The choice of the number of pools willamong others depend on (I) the interval shift δp induced by the chemicaland/or enzymatic alteration, ii) the elution window of the fractionscollected from the primary chromatographic separation and iii) the needto optimise the chromatography time and the analysis time. The currentinvention also provides the use of a parallel column sorter. With aparallel column sorter, the method based on a single column is executedwith a number of columns operating in parallel (i.e., synchronously).The parallel sorter contains a number of identical columns which are runin exactly the same conditions (flow rate, gradient, etc.). A parallelcolumn sorter is most conveniently a device where 2, 3, 4 or morecolumns perform a secondary chromatographic run at the same time insubstantially similar conditions (flow rate, gradient, etc.) and whereinthe exit of the parallel sorter is directly connected with an analyzer.A parallel column sorter divides the chromatographic separation timewhich is normally needed for a series of serial single columns byapproximately the number of columns which are used in said parallelsorter. The advantage of using a parallel column sorter is not only thatthe overall compound-peptide complexes sorting time can be significantlyreduced, but also that there are a limited number of dead intervalsbetween the selection of altered compound-peptide complexes from thealtered fractions so that the detection of the altered compound-peptidecomplexes can occur in a continuous manner. In another aspect of theinvention, a multi-column peptide sorter can be used. Such amulti-column peptide sorter is created and essentially exists of anumber of parallel column sorters that are operating in a combinedparallel and serial mode. Such parallel sorter essentially comprises ytimes a set of z columns, wherein the z columns are connected inparallel. In a non-limiting example, a multi-column sorter where y=3 andz=3 is a nine-column sorter. Such a nine-column sorter operates withthree sets of each time three columns connected in parallel. The threeparallel column sets are designated as A, B, and C. The individualcolumns of A are designated as I, II, and III; the individual columns ofB are designated as I′, II′; and III′; and the individual columns of Care designated as I″, II″ and III″. One set of parallel columns operateswith a delay (named θ) versus the previous set. Therefore, the parallelsorter B starts with a delay of θ with respect to the parallel sorter A,and the parallel sorter C starts with a delay of θ after the start ofthe parallel sorter B, and with a delay of 2θ after the start of theparallel sorter A. It is important to note that in the multi-columnsorter, only one run 1 fraction of altered compound-peptide complexes isprocessed at a given time per column. Thus, in the example of anine-column sorter, nine fractions of altered compound-peptide complexesare processed simultaneously. This differs from the two previousdescribed sorters (i.e., a one column peptide sorter and a parallelsorter) where several altered fractions are strategically pooled andloaded simultaneously. As only one fraction of altered compound-peptidecomplexes is processed at the time on the multi-column sorter, thecontrol of the flow rate accuracy (i.e., in the secondarychromatographic step) is not as important as in the previous sorters.Another advantage of the multi-column sorter is that it is well adaptedto separate altered compound-peptide complexes from non-alteredcomplexes in cases where the chromatographic shift of alteredcompound-peptide complexes varies significantly throughout the differentfractions. It will be clear to those skilled in the art that othercombinations of parallel and serial columns can lead to similar results.The choice of the number of columns, their arrangement and the fractionsloaded on the columns will among others depend on i) the interval δpinduced by the chemical or enzymatic alteration, ii) the elution windowof the fractions collected from the primary chromatographic separationand iii) the need to optimise the chromatography time and the analysistime. It will further be clear to a person skilled in the art thatpeptide sorters that carry out the method of the current invention couldalso be performed in a fully automated manner, using commerciallyavailable auto-injectors, HPLC-equipment and automated fractioncollectors. Therefore, the present examples of peptide sorters shouldnot be considered as exhaustive. Several variants, includingelectrophoretic and ion-exchange chromatography systems, are equallyfeasible. The illustrative embodiment further provides a system forperforming the above-described method of proteome analysis in aselective and efficient manner. As discussed, a primary chromatographiccolumn performs an initial separation of the complex peptide mixture.The primary chromatographic column separates the complex peptide mixtureinto at least two fractions under a defined set of conditions. Forexample, the primary chromatographic column separates the proteinpeptide mixture by eluting the column with a predetermined solventgradient and a predetermined flow rate. The fractions resulting from theprimary chromatographic separation may be strategically pooled tocombine a plurality of fractions having distinct elution times into aplurality of pooled fractions, as described above. The pooled fractionsmay be subsequently altered to result in a set of altered peptides and aset of non-altered peptides for each fraction. According to an alternateembodiment, the fractions are first altered using the methods describedabove and then strategically pooled into a set of pooled fractions,wherein each fraction in a pooled fraction comprises a set of alteredcompound-peptide complexes and a set of non-altered compound-peptidecomplexes. In a secondary chromatographic separation, the alteredcomplexes are separated from the unaltered complexes. The isolatedtargets (=the altered compound-peptide complexes) may then be analyzedto identify a protein.

In another embodiment the present invention further provides a method toidentify the isolated targets (=the altered compound-target complexes).In a particular embodiment the identification of the targets can becarried out by a mass spectrometric approach. In another particularembodiment where the target molecules are proteins or peptides saididentifying step is performed by a method selected from the groupconsisting of: a tandem mass spectrometric method, Post-Source Decayanalysis, measurement of the mass of the peptides, in combination withdatabase searching. In yet another particular embodiment theidentification method based on the mass measurement of the peptides isfurther based on one or more of the following: (a) the determination ofthe number of free amino groups in the target peptides; (c) theknowledge about the cleavage specificity of the protease used togenerate the protein peptide mixture; and (d) the grand average of thehydropathicity of the target peptides.

In a particular embodiment the targets are proteins or peptides andtherefore the method of the invention is further coupled to a peptideanalysis. The present invention therefore further provides a method toidentify target peptides and their corresponding proteins. In apreferred approach the analysis of altered drug-peptide complexes isperformed with a mass spectrometer. However, drug-peptide complexes canalso be further analysed and identified using other methods such aselectrophoresis, activity measurement in assays, analysis with specificantibodies, Edman sequencing, etc. An analysis or identification stepcan be carried out in different ways. In one way, altered drug-peptidecomplexes (the tagged peptides) eluting from the chromatographic columnsare immediately directed to the analyzer. In an alternative approach,altered drug-peptide complexes are collected in fractions. Suchfractions may or may not be manipulated before going into furtheranalysis or identification. An example of such manipulation consists ofa concentration step, followed by spotting each concentrate on forinstance a MALDI-target for further analysis and identification. In apreferred embodiment altered drug-peptide complexes are analysed withhigh-throughput mass spectrometric techniques.

The information obtained is primarily the mass of the tagged peptide(s).This mass is the sum of the mass of the peptide and the mass of the tag(the altered compound component). Since the latter mass is known fromthe alteration reaction, this tag mass can be subtracted from the totalmass of the tagged peptide resulting in a peptide mass which will be thebasis in further searching algorithms.

Generally, a mass information is not sufficient for unambiguous peptideidentification. Therefore the tagged peptides (=the alteredcompound-peptide complexes) are further fragmented. This is often doneby collision-induced dissociation (CID) in an electrospray instrument orMALDI and is generally referred to as MS/MS or tandem mass spectrometry.Manual or automated interpretation of these MS/MS spectra leads to theassignment of sequence tags and to the identification of the peptidesequence tags and to the location of the tag. Protein identificationsoftware which can be used in the present invention to compare theexperimental fragmentation spectra of the tagged peptide with amino acidsequences stored in peptide databases. Such algorithms are available inthe art.

One such algorithm, ProFound, uses a Bayesian algorithm to searchprotein or DNA database to identify the optimum match between theexperimental data and the protein in the database. ProFound may beaccessed on the World-Wide Web at <http//prowl.rockefeller.edu> and<http//www.proteometrics.com>. Profound accesses the non-redundantdatabase (NR). Peptide Search can be accessed at the EMBL website. Seealso, Chaurand P. et al. (1999) J. Am. Soc. Mass. Spectrom 10, 91,Patterson S. D., (2000), Am. Physiol. Soc., 59-65, Yates J R (1998)Electrophoresis, 19, 893). MS/MS spectra may also be analysed by MASCOT(available at http://www.matrixscience.com, Matrix Science Ltd. London).

Any mass spectrometer may be used to analyze the altered drug-peptidecomplexes. Non-limiting examples of mass spectrometers include thematrix-assisted laser desorption/ionization (“MALDI”) time-of-flight(“TOF”) mass spectrometer MS or MALDI-TOF-MS, available from PerSeptiveBiosystems, Framingham, Mass.; the Ettan MALDI-TOF from AP Biotech andthe Reflex III from Brucker-Daltonias, Bremen, Germany for use inpost-source decay analysis; the Electrospray Ionization (ESI) ion trapmass spectrometer, available from Finnigan MAT, San Jose, Calif.; theESI quadrupole mass spectrometer, available from Finnigan MAT or theQSTAR Pulsar Hybrid LC/MS/MS system of Applied Biosystems Group, FosterCity, Calif. and a Fourrier transform mass spectrometer (FTMS) using aninternal calibration procedure (O'Connor and Costello (2000) Anal. Chem.72, 5881-5885).

Alternatively, tagged peptide ions can decay during their flight afterbeing volatilised and ionised in a MALDI-TOF-MS. This process is calledpost-source-decay (PSD). Knowing the peptide sequences stored in peptidesequence databases, it is possible to deduce parts of or the totalsequence from such PSD spectra. As above, this analysis can be donemanually or by using computer algorithms which are well known in thefield. One such algorithm is for instance the MASCOT program.

In a particular embodiment additional sequence information can beobtained in MALDI-PSD analysis when the alfa-NH₂-terminus of the targetpeptides is altered with a sulfonic acid moiety group. Target peptidescarrying an NH₂-terminal sulfonic acid group are induced to particularfragmentation patterns when detected in the MALDI-TOF-MS mode. Thelatter allows a very fast and easy deduction of the amino acid sequence.

Alternatively, tagged peptides could also be analyzed by conventionalEdman-degradation and the obtained amino acid sequence compared tosequences stored in protein or genomic sequence databases. In case thecompound itself is a peptide, then Edman-sequencing will generate ateach cycle a double amino acid identification, until the degradationreaches the residue of one of the chains which is involved in theisopeptide linkage.

Once, a tagged peptide is unambiguously identified by MS-basedfragmentation analysis, further similar experiments may then simply useits total mass. This is for instance the case when activity-basedprotein profiling of a specific target is carried out on a large numberof samples. Indeed, the amount of tagged peptide formed will bedependent on the accessibility and specific reactivity of the target.Once the specific tagged peptide fully characterized in terms of totalmass and elution times, it suffices to select the tagged peptide basedon its exact mass. A peptide mass can be sufficiently accuratelymeasured with a Fourrier transform mass spectrometer (FT-MS) or usingrecently developed MALDI based time of flight machines. Such machinesare for instance constructed by Bruker-Daltonics, Bremen, Germany(Ultraflex).

If the accuracy by which the mass of the tagged peptide can be measuredis not sufficiently discriminative, then additional information can begenerated. For example, the elution time by which a given peptide elutesduring chromatography, is a parameter which is totally independent ofthe peptide mass.

Thus the probability is low that two or more peptides, with identicalmasses or with masses falling within the error range of the massmeasurements, also elute with identical or very similar retention timesduring chromatography. Since the retention time of a peptide duringRP-chromatography is primarily related to its overall hydrophobicity,the Grand Average hydropathicity (GRAVY) index, which can be calculatedusing hydropathicity values given to every natural amino acid. Thus themass together with the GRAVY index are two independent parameters highlycharacteristic for a given peptide.

In another embodiment the method of determining the identity of theparent protein by only accurately measuring the peptide mass of at leastone target peptide can be improved by further enriching the informationcontent of the selected target peptides. As a non-limiting example ofhow information can be added to the target peptides, the free NH₂-groupsof these peptides can be specifically chemically changed in a chemicalreaction by the addition of two different isotopically labelled groups.As a result of this change, said peptides acquire a predetermined numberof labelled groups. Since the change agent is a mixture of twochemically identical but isotopically different agents, the targetpeptides are revealed as peptide twins in the mass spectra. The extentof mass shift between these peptide doublets is indicative for thenumber of free amino groups present in said peptide. To illustrate thisfurther, for example the information content of target peptides can beenriched by specifically changing free NH₂-groups in the peptides usingan equimolar mixture of acetic acid N-hydroxysuccinimide ester andtrideuteroacetic acid N-hydroxysuccinimide ester. As the result of thisconversion reaction, peptides acquire a predetermined number of CH₃—CO(CD₃-CO) groups, which can be easily deduced from the extent of theobserved mass shift in the peptide doublets. As such, a shift of 3 amu'scorresponds with one NH₂-group, a 3 and 6 amu's shift corresponds withtwo NH₂-groups and a shift of 3, 6 and 9 amu's reveals the presence ofthree NH₂-groups in the peptide. This information further supplementsthe data regarding the peptide mass and/or the knowledge that thepeptide was generated with a protease with known specificity.

The use of the mass of a sorted tagged peptide as the solepeptide/protein identification criterion becomes important andreasonable once said tagged-peptide has been fully identified(previously) by other means such as those described above.

For instance, once a tagged peptide has been fully identified byMS-fragmentation analysis and database searching, further identificationcan be based on the accurately measured mass of the tagged peptide,without repeating each time the MS/MS-analysis.

Thus the expression levels or the activity and expression levels of abiological target or different biological targets present in a multitudeof samples means more than one, preferably more than five, morepreferably more than one hundred and more preferably more than thousandand more preferably a number typically encountered duringhigh-throughput analysis. A highly complex mixture of proteins refers tocell lysates, cell fractions, tissues, biological fluids and the like asthey are described below.

In cases where the invention leads to the identification of the membersof a class of biological targets, then the mass of every tagged peptidecould be representative of its corresponding biological target proteinand the invention would allow a global analysis of levels or levelsand/or activities of each member of the family. For instance, the use ofFSBA to target ATP-binding proteins and in particular the kinasefamilies, can lead to a number of tagged peptides. Each of these taggedpeptides will carry the same tag but will be otherwise distinct by thepeptide-moiety. Thus each kinase level and/or activity will be reflectedby the specific peptide mass in the tagged peptide. Relativequantification of every tagged peptide will provide a global profile oflevels and/or activities of the members of a family of biologicaltargets.

Although absolute quantification of peptides by mass spectrometry isvery difficult, MS-based techniques are suitable for comparativeanalysis.

Thus in another embodiment a method is provided to determine therelative amount of the level and/or activity of at least one targetprotein in more than one sample comprising proteins, comprising thesteps of (a) the addition of a compound comprising a first isotope to afirst sample comprising peptides wherein said compound stably interactswith at least one peptide forming a compound-peptide complex; (b) theaddition of a compound comprising a second isotope to a second samplecomprising peptides wherein said compound stably interacts with at leastone peptide forming a compound-peptide complex; (c) combining theprotein peptide mixture of the first sample with the protein peptidemixture of the second sample; (d) separating the combined proteinpeptide mixtures into fractions of peptides via chromatography; (e)chemically, or enzymatically, or chemically and enzymatically, alteringsaid compound present on at least one compound-peptide complex in eachfraction; (f) isolating the altered compound-peptide complexes out ofeach fraction via chromatography, wherein the chromatography isperformed with the same type of chromatography as in step (d); (g)performing mass spectrometric analysis of the isolated alteredcompound-peptide complexes; (h) calculating the relative amounts of thealtered compound-peptide complexes in each sample by comparing the peakheights of the identical but differentially, isotopically labelledaltered compound-peptide complexes, and (i) determining the identity ofsaid peptides in the altered compound-peptide complexes and theircorresponding proteins.

To compare the level and/or activity of the targets in two differentsamples, differential mass labeling can be used. Therefore, thecompound-peptide complexes (the tagged peptides) of the first sample canbe labeled with “light” atoms, while the tagged peptide of the secondsample will be labeled with “heavy atoms”. Labeling can for instance becarried out by the use of a compound that carries an isotopic label.Before the primary chromatographic run the compound-peptide targetcomplexes of both samples will be mixed. The “light” and “heavy”components will elute or migrate in an identical or nearly identicalmanner during the primary run. Their alteration will also proceed in thesame manner. The “light” and “heavy” tagged peptides will elute ormigrate in an identical or nearly identical manner and co-transferred tothe mass spectrometer. Only during the analysis with the latter, “light”and “heavy” tagged peptide ions will segregate and their relativeintensities can be measured. It is important to stress that thediscriminating atoms remain attached to the tagged peptide after thealteration step. Thus both the “light” and “heavy” atoms are part of thetag on the tagged peptide.

As couple of light and heavy atoms, one can use H/D, ¹⁶O/¹⁸O, ¹²C/¹³C,¹⁴N/¹⁵N or any couple of stable isotopes which can be stablyincorporated in organic and inorganic compounds. In an alternativeembodiment the proteins can be labeled instead of the compounds. Thedifferential isotopic labeling of peptides in a first and a secondsample can be done in many different ways available in the art. A keyelement is that a particular peptide originating from the same proteinin a first and a second sample is identical, except for the presence ofa different isotope in one or more amino acids of the peptide. In atypical embodiment the isotope in a first sample will be the naturalisotope, referring to the isotope that is predominantly present innature, and the isotope in a second sample will be a less commonisotope, hereinafter referred to as an uncommon isotope. Examples ofpairs of natural and uncommon isotopes are H and D, O¹⁶ and O¹⁸, C¹² andC¹³, N¹⁴ and N¹⁵. Peptides labeled with the heaviest isotope of anisotopic pair are herein also referred to as heavy peptides. Peptideslabeled with the lightest isotope of an isotope pair are herein alsoreferred to as light peptides. For instance, a peptide labeled with H iscalled the light peptide, while the same peptide labeled with D iscalled the heavy peptide. Peptides labeled with a natural isotope andits counterparts labeled with an uncommon isotope are chemically verysimilar, separate chromatographically in the same manner and also ionizein the same way. However, when the peptides are fed into an analyser,such as a mass spectrometer, they will segregate into the light and theheavy peptide. The heavy peptide has a slightly higher mass due to thehigher weight of the incorporated, chosen isotopic label. Because of theminor difference between the masses of the differentially isotopicallylabeled peptides the results of the mass spectrometric analysis ofisolated altered compound-peptide complexes will be a plurality of pairsof closely spaced twin peaks, each twin peak representing a heavy and alight altered complex. Each of the heavy complexes is originating fromthe sample labelled with the heavy isotope; each of the light complexesis originating from the sample labelled with the light isotope. Theratios (relative abundance) of the peak intensities of the heavy and thelight peak in each pair are then measured. These ratios give a measureof the relative amount (differential occurrence) of that target (as anisolated altered compound-complex) in each sample. The peak intensitiescan be calculated in a conventional manner (e.g. by calculating the peakheight or peak surface). As herein described above, the alteredcompound-peptide complexes can also be identified allowing theidentification of proteins in the samples. If a protein target for aparticular compound is present in one sample but not in another, theisolated altered compound-peptide complexes (corresponding with thisprotein) will be detected as one peak which can either contain the heavyor light isotope. However, in some cases it can be difficult todetermine which sample generated the single peak observed during massspectrometric analysis of the combined sample. This problem can besolved by double labeling the first sample, either before or after theproteolytic cleavage, with two different isotopes or with two differentnumbers of heavy isotopes. Examples of labeling agents are acylatingagents.

Incorporation of the natural and/or uncommon isotope in peptides can beobtained in multiple ways. In one approach proteins are labeled in thecells. Cells for a first sample are for instance grown in mediasupplemented with an amino acid containing the natural isotope and cellsfor a second sample are grown in media supplemented with an amino acidcontaining the uncommon isotope. In another embodiment the incorporationof the differential isotopes can also be obtained by an enzymaticapproach. For instance labeling can be carried out by treating onesample comprising proteins with trypsin in “normal” water (H₂ ¹⁶O) andthe second sample comprising proteins with trypsin in “heavy” water (H₂¹⁸O). As used herein “heavy water” refers to a water molecule in whichthe O-atom is the ¹⁸O-isotope. Trypsin shows the well-known property ofincorporating two oxygens of water at the COOH-termini of the newlygenerated sites. Thus in sample one, which has been trypsinized in H₂¹⁶O, peptides have “normal” masses, while in sample two, peptides(except for most of the COOH-terminal peptides) have a mass increase of4 amu's corresponding with the incorporation of two 180 atoms Thisdifference of 4 amu's is sufficient to distinguish the heavy and lightversion of the altered compound-peptide complexes in a mass spectrometerand to accurately measure the ratios of the light versus the heavypeptides and thus to determine the ratio of the corresponding targetpeptides/target proteins in the two samples.

Incorporation of the differential isotopes can further be obtained withmultiple labelling procedures based on known chemical reactions that canbe carried out at the protein or the peptide level. For example,proteins can be changed by the guadinylation reaction withO-methylisourea, converting NH₂-groups into guanidinium groups, thusgenerating homoarginine at each previous lysine position. Proteins froma first sample can be reacted with a reagent with the natural isotopesand proteins from a second sample can be reacted with a reagent with anuncommon isotope. Peptides could also be changed by Shiff's-baseformation with deuterated acetaldehyde followed by reduction with normalor deuterated sodiumborohydride. This reaction, which is known toproceed in mild conditions, may lead to the incorporation of apredictable number of deuterium atoms. Peptides will be changed eitherat the α-NH₂-group, or ε-NH₂ groups of lysines or on both. Similarchanges may be carried out with deuterated formaldehyde followed byreduction with deuterated NaBD₄, which will generate a methylated formof the amino groups. The reaction with formaldehyde could be carried outeither on the total protein, incorporating deuterium only at lysine sidechains or on the peptide mixture, where both the α-NH₂ andlysine-derived NH₂-groups will be labelled. Since arginine is notreacting, this also provides a method to distinguish between Arg- andLys-containing peptides. Primary amino groups are easily acylated with,for example, acetyl N-hydroxysuccinimide (ANHS). Thus, one sample can beacetylated with normal ANHS whereas a second sample can be acylated witheither ¹³CH₃CO—NHS or CD₃CO—NHS. Also the ε-NH₂ group of all lysines isin this way derivatized in addition to the amino-terminus of thepeptide. Still other labelling methods are for example acetic anhydridewhich can be used to acetylate hydroxyl groups and trimethylchlorosilanewhich can be used for less specific labelling of functional groupsincluding hydroxyl groups and amines.

In yet another approach the primary amino acids are labelled withchemical groups allowing to differentiate between the heavy and thelight peptides by 5 amu, by 6 amu, by 7 amu, by 8 amu or even by largermass difference. Alternatively, the differential isotopic labelling iscarried out at the carboxy-terminal end of the peptides, allowing thedifferentiation between the heavy and light variants by more than 5 amu,6 amu, 7 amu, 8 amu or even larger mass differences. Since the methodsof the present invention do not require any prior knowledge of the typeof target proteins that may be present in the samples, they can be usedto determine the relative amounts of both known and unknown targetproteins which are present in the samples examined.

The methods provided in the present invention to determine relativeamounts of at least one protein target and/or the activity of a proteinin at least two samples can be broadly applied to compare protein levelsin for instance cells, tissues, or biological fluids, organs, and/orcomplete organisms. Such a comparison includes evaluating subcellularfractions, cells, tissues, fluids, organs, and/or complete organismswhich are, for example, diseased and non-diseased, stressed andnon-stressed, drug-treated and non drug-treated, benign and malignant,adherent and non-adherent, infected and uninfected, transformed anduntransformed. The method also allows to compare protein target levelsor the activity of one or more proteins in subcellular fractions, cells,tissues, fluids, organisms, complete organisms exposed to differentstimuli or in different stages of development or in conditions where oneor more genes are silenced or overexpressed or in conditions where oneor more genes have been knocked-out.

In another embodiment, the methods described herein can also be employedin diagnostic assays for the detection of the presence, the absence or avariation in level of one or more protein targets and/or the activity ofa protein or a specific set of proteins indicative of a disease state(e.g., such as cancer, neurodegenerative disease, inflammation,cardiovascular diseases, viral infections, bacterial infections, fungalinfections or any other disease). Specific applications include theidentification of target proteins which are present in metastatic andinvasive cancers, the differential expression of proteins in transgenicmice, the identification of proteins that are up- or down-regulated indiseased tissues, the identification of intracellular changes in cellswith physiological changes such as metabolic shift, the identificationof biomarkers in cancers, the identification of signalling pathways.

Samples that can be analyzed by methods of the invention includebiological samples, such as cell lysates, microsomal fractions, cellfractions, tissues, organelles, etc., and biological fluids includingurine, sputum, saliva, synovial fluid, nipple aspiration fluid, amnionfluid, blood, cerebrospinal fluid, tears, ejaculate, serum, pleuralfluid, ascites fluid, stool, or a biopsy sample. If the sample is impure(e.g., plasma, serum, stool, ejaculate, sputum, saliva, cerebrospinalfluid, or blood or a sample embedded in paraffin), it may be treatedprior to employing a method of the invention, frequently to removecontaminants of the components of interest. Procedures include, forexample, filtration, extraction, centrifugation, affinity sequestering,etc. Where the probes do not readily pass through a cellular membrane,intact or permeabilized, or where a lysate is desirable, the cells aretreated with a reagent effective for lysing the cells contained in thefluids, tissues, or animal cell membranes of the sample, and forexposing the proteins contained therein and, as appropriate, partiallyseparating the proteins from other aggregates or compounds such asmicrosomes, lipids, carbohydrates and nucleic acids in the sample.Methods for purifying or partially purifying proteins from a sample arewell known in the art (e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, 1989, herein incorporatedby reference). The samples may come from different sources and be usedfor different purposes.

Usually, a proteome will be analyzed. By a proteome is intended at leastabout 20% of total protein coming from a biological sample source,usually at least about 40%, more usually at least about 75%, andgenerally 90% or more, up to and including all of the protein obtainablefrom the source. Thus the proteome may be present in an intact cell, alysate, a microsomal fraction, an organelle, a partially extractedlysate, biological fluid, and the like. The proteome will be a complexmixture of proteins, generally having at least about 20 differentproteins, usually at least about 50 different proteins and in most cases100 different proteins or more. In effect, the proteome is a complexmixture of proteins from a natural source and will usually involvehaving the potential of having 10, usually 20, or more proteins that aretarget proteins for a specific compound used to analyze the proteomeprofile. The sample will be representative of the target proteins ofinterest. The sample may be adjusted to the appropriate bufferconcentration and pH, if desired. One or more compounds, having thestructure SLA, may then be added, each at a concentration in the rangeof about 0.001 mM to 20 mM. After incubating the reaction, generally fora time for the reaction to go substantially to completion, generally forabout 1-60 min, at a temperature in the range of about 20-40° C., thereaction may be quenched.

The method of the present invention is useful in supporting thedevelopment of new drugs and identifying (new) drug targets. Oneembodiment of the subject invention is especially useful for rapidlyscreening a number of drug candidate compounds. The invention is alsouseful for systematically analyzing a number of compounds that may varygreatly in their chemical structure or composition, or that may vary inminor aspects of their chemical structure or composition. The inventionis also useful for optimizing candidate drugs that show the mostmedicinal promise, meaning binding to a particular, desired target andnot to others. The invention can also be used to measure enzymaticactivities or biological activities in general or the sum of expressionlevels and activities of biological molecules in total extracts oftissues, cells, cell organelle and protein complexes. The ability topredict the toxic effects of potential new drugs is crucial toprioritizing compound pipelines and eliminating costly failures in drugdevelopment. Toxicogenomics, which deals primarily with the effects ofcompounds on gene expression patterns in target cells or tissues, isemerging as a key approach in screening new drug candidates because itmay reveal genetic signatures that can be used to predict toxicity inthese compounds. The current invention focuses on a proteomic approachfor the detection of drug targets and hence the method could bedesignated as toxicoproteomics. The method of the present inventioncould also be used for the design and optimization of clinical trials.With the method is possible to develop potentially, smaller clinicaltrials targeting more specific populations that are likely to respond tothe drug and that are not likely to develop adverse drug reactions.This, in turn, the use of the method could potentially reduce the costand time required for clinical trials.

In what follows, a more informative description of several of thedifferent steps of the invention is presented.

I. Preparation of a Protein Peptide Mixture

Protein peptide mixtures originating from a sample comprising proteinsfor a compound treated sample comprising proteins are obtained bymethods described in the art such as chemical or enzymatic cleavage ordigestion. In a preferred aspect, the proteins and compound-proteincomplexes are digested by a proteolytic enzyme. Trypsin is aparticularly preferred enzyme because it cleaves at the sites of lysineand arginine, yielding charged peptides which typically have a lengthfrom about 5 to 50 amino acids and a molecular weight of between about500 to 5,000 dalton. Such peptides are particularly appropriate foranalysis by mass spectroscopy. A non-limited list of proteases which mayalso be used in this invention includes Lysobacter enzymogenesendoproteinase Lys-C, Staphylococcus aureus endoproteinase Glu-C (V8protease), Pseudomonas fragi endoproteinase Asp-N and clostripain.Proteases with lower specificity such as Bacillus subtilis subtilisin,procain pepsin and Tritirachium album proteinase K may also be used inthis invention.

Alternatively, chemical reagents may also be used to cleave the proteinsinto peptides. For example, cyanogen bromide may be used to cleaveproteins into peptides at methionine residues. Chemical fragmentationcan also be applied by limited hydrolysis under acidic conditions.Alternatively, BNPS-skatole may be used to cleave at the site oftryptophan. Partial NH₂-terminal degradation either using chemicallyinduced ladders with isothiocyanate or using aminopeptidase treatmentcan be used as well.

II. Chromatography

As used herein, the term “chromatographic step” or chromatography”refers to methods for separating chemical substances and are vastlyavailable in the art. In a preferred approach it makes use of therelative rates at which chemical substances are adsorbed from a movingstream of gas or liquid on a stationary substance, which is usually afinely divided solid, a sheet of filter material, or a thin film of aliquid on the surface of a solid. Chromatography is a versatile methodthat can separate mixtures of molecules even in the absence of detailedprevious knowledge of the number, nature, or relative amounts of theindividual substances present. The method is widely used for theseparation of chemical molecules of biological origin (for example,amino acids, fragments of proteins, peptides, proteins, phospholipids,steroids etc.) and of complex mixtures of petroleum and volatilearomatic mixtures, such as perfumes and flavours. The most widely usedcolumnar liquid technique is high-performance liquid chromatography, inwhich a pump forces the liquid mobile phase through a high-efficiency,tightly packed column at high pressure. Recent overviews ofchromatographic techniques are described by Meyer M., 1998, ISBN:047198373X and Cappiello A. et al. (2001) Mass Spectrom. Rev. 20(2):88-104, incorporated herein by reference. Other recently developedmethods described in the art and novel chromatographic methods comingavailable in the art can also be used. Some examples of chromatographyare reversed phase chromatography (RP), ion exchange chromatography,hydrophobic interaction chromatography, size exclusion chromatography,gel filtration chromatography or affinity chromatography such asimmunoaffinity and immobilized metal affinity chromatography.

Chromatography is one of several separation techniques. Electrophoresisand all variants such as capillary electrophoresis, free flowelectrophoresis etc. is another member of this group. In the lattercase, the driving force is an electric field, which exerts differentforces on solutes of different ionic charge. The resistive force is theviscosity of the non-flowing solvent. The combination of these forcesyields ion mobilities peculiar to each solute. Some examples are sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and nativegel electrophoresis. Capillary electrophoresis methods include capillarygel electrophoresis, capillary zone electrophoresis, capillaryelectrochromatography, capillary isoelectric focussing and affinityelectrophoresis. These techniques are described in McKay P., AnIntroduction to Chemistry, Science Seminar, Department of RecoverySciences, Genentech, Inc. incorporated herein by reference.

III. Buffers

The methods of the invention require compatibility between theseparation conditions in the primary run, the reaction conditions in thealteration step, the separation condition in the secondary run and theconditions to analyse the eluting altered compound-peptide complexes inanalysers such as mass spectrometers. As mentioned before, thecombination of the chromatographic conditions in the primary andsecondary run and the chromatographic shifts induced by the alterationreaction is determining the possibility to isolate the alteredcompound-peptide complexes out of each fraction obtained from a proteinpeptide mixture in the primary run. As also mentioned before, in apreferred embodiment the chromatographic conditions of the primary runand the secondary run are the same or substantially similar.

In a further preferred embodiment, buffers and or solvents used in bothchromatographic steps are compatible with the conditions required toallow an efficient proceeding of the chemical and/or enzymatic reactionsin the alteration step in between the two chromatographic steps. In aparticular preferred embodiment the nature of the solvents and buffer inthe primary run, the secondary run and the alteration step are identicalor substantially similar. In a further preferred embodiment said buffersand solvents are compatible with the conditions required to perform amass spectrometric analysis. Defining such buffers and solvents needstuning and fine-tuning [and such conditions are not available in theprior art].

For some embodiments of the invention with particular types of alteredcompound-peptide complexes it is very difficult if not impossible todesign one set of identical or substantially similar buffers and/orsolvents which can be used throughout the procedure of primary run,alteration step, secondary run and analysis.

For instance, the chemical and/or enzymatic reaction to alter thecompound-peptide complexes in the alteration step may request specificreaction conditions which are not compatible with the buffers used inthe primary and/or secondary run. In these cases the buffer/solventconditions in the fractions are changed before the alteration stepand/or after the alteration step which changing is performed withmethods described in the art such as for example an extraction, alyophilisation and redisolving step, a precipitation and redisolvingstep, a dialysis against an appropriate buffer/solvent or even a fastreverse phase separation with a steep gradient.

Another complication may be the composition of the buffer/solventpresent in a complex protein mixture or a protein peptide mixture beforestarting the primary run. Application of a pre-treatment step mayrequest specific buffer/solvent conditions which are not compatible withthe buffer/solvent to perform the primary run. Alternatively, theconditions for the preparation/isolation of proteins from theirbiological source may result in the contamination of the proteinmixtures or protein peptide mixtures with compounds which negativelyinterfere with the compound reaction and/or with the primary run. Inthese situations the buffer/solvent composition of the protein mixtureor the protein peptide mixture is changed to make them compatible withthe primary run. Such changing is performed with methods described inthe art such as for example an extraction, a lyophilisation andredisolving step, a precipitation and redisolving step, a dialysisagainst an appropriate buffer/solvent or even a fast reverse phaseseparation with a steep gradient.

In yet another embodiment of the invention the buffer/solvent of thesecondary run is not compatible with performing the analysis of theeluting altered compound-peptide complexes. In such cases, thebuffer/solvent in the fractions collected from the secondary run ischanged to make the conditions compatible with the analysis with forinstance a mass spectrometer. Such changing is performed with methodsdescribed in the art such as for example an extraction, a lyophilisationand redisolving step, a precipitation and redisolving step, a dialysisagainst an appropriate buffer/solvent or even a fast reverse phaseseparation with a steep gradient. Alternatively, the fractions with thealtered compound-peptide complexes can be collected and recombined for athird series of separations, hereinafter referred to as a ternary run.Said ternary run is designed in such a way that the eluting flagged oridentification peptides can be analysed with a mass spectrometer.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. For examplechromatography can be substituted in many cases by electrophoresis.Electrophoretic techniques include (capillary) gel electrophoresis,(capillary) electrochromatography, (capillary) isoelectric focussing andaffinity electrophoresis.

EXAMPLES

1. The Identification of Drug Targets

1.1 In a particular compound alll three properties “SLA” reside in thesame moiety.For instance the compound benzoyl-penicilline forms an acyl-enzymeadduct with its target the bacterial DD-aminotranspeptidase. Afterproteolytic cleavage a penicilloyl-peptide is generated. The alterationstep may consist in a conversion of the thioether into a sulfoxidederivative which is more hydrophilic and separates distinctly duringchromatographic run 2.

1.2. In a particular compound the “SLA”-moieties are partiallyseparated. The “S”-part interacts with the target molecule. Chemicalcross-linking is established by the same group. Thus “S” and “L” are thesame. The third “A”-group is altered.

For instance, the molecule could be composed of a Lys-containing peptidewhich can be cross-linked to Gln-41 of G-actin, through the catalyticaction of a transglutaminase such as factor XIIIa (specific labelling ofG-actin at Gln-41 with cadaverine or cadaverine-derivatives byzero-length cross-linking with a transglutaminase has been reportedpreviously (Takashi (1988) Biochemistry 27(3): 938). Theisopeptide-linkage created between Gln-41 of actin and theLys-containing peptide is similar.

The sequence of the compound peptide in the one-letter notation isAc-F-I-E-G-R-A-D-S-K-S-S-COOH has an acetylated free α-NH₂-terminus anda free COOH-terminus. According to our “SLA”-definitions, we distinguishthe following functions:

The specificity-determining group (“S”) is composed of the Lys-residue,flanked on both sides by Ser-residues. The Ser-residues and the Asp,incorporated in the COOH-terminal part, further contribute to thehydrophilic character (and therefore solubility) of the finalcross-linked peptide. They also contrast with the hydrophobic Phe-Ilecluster, located in the extreme NH₂-terminus, forming ahydrophobic-hydrophilic balance, which will be broken during thealteration step.

The Lys-residue, determining the transglutaminase reaction specificity,is also the residue involved in the zero-length isopeptide formation.Thus here “S” and “L” are the same moieties.

The factor Xa-restriction cleavage site, forms the “A”-part of thecompound and is spatially separated from the “S-L”-part. When releasedby cleavage, the hydrophobic Ac-F-I-E-G-R cargo will separate, leaving amore hydrophilic compound still attached to its target peptide. In thesecondary run (run 2), this more hydrophilic peptide will shift in frontof the bulk of unmodified peptides.

This experiment will be described in detail in examples 1.5 and 1.61.3 In a particular compound the three properties “SLA” are moreseparated: the specificity determining group “S” interacts with thetarget molecule, chemical cross-linking (“L”) is established by a secondgroup while a third moiety (“A”) is subject to alteration. The resultingtagged peptide still carries the “S” or part of the “S” moiety.

For instance, the molecule could be composed of the caspase-1 inhibitorypeptide aldehyde Ac-YVAD-CHO, elongated versus the N-terminal side by, ashort peptide carrying the factor X_(a) restriction cleavage site, forinstance:

Ac-A-A-I-E-G-R-Y-V-A-D-CHO. While the Y-V-A-D-sequence will direct themolecule to the active site of the caspase-1 type proteases (“S”-group)the COOH-terminus converted into an aldehyde will create the cross-link(“L”-group). The NH₂-terminal part of the molecule can be cleaved off byusing factor X_(a).

1.4 In one compound the three properties “SLA” are more separated.

The specificity-determining group “S” interacts with the targetmolecule. Cross-linking is established by another moiety in the moleculereacting with another part of the target. The alteration consists in theseparation of the “S”- and the “L”-moieties. The tag now does notcontain the “S”-group anymore but the “L”-group or part of the“L”-group.

For instance fluorosulfenylbenzoyl adenosine FSBA can form a complexwith ATP-binding proteins. FSBA reacts covalently with a lysinederivative located in the active center opposite to the interaction siteof the adenosyl-moiety. The FSBA-peptide complex is generated uponproteolytic cleavage. The alteration step consists of analkaline-induced hydrolysis of the molecule leaving only thesulfenylbenzoyl moiety attached as tag on the peptide. Since FSBA isknown to mimic ATP-binding, this method could be used to localize theATP-binding site(s) in the primary structure of target proteins, toidentify ATP-binding proteins and to profile kinase activities in aglobal cellular context.

1.5 Identification of the Target Site of a Compound in a PurifiedProtein

In this example, purified skeletal muscle actin was covalently linkedwith a synthetic Lys-containing peptide at the actin Gln-41 position.The design and sequence of the synthetic peptide, here referred as“compound peptide or CP” is described in example 1.2.

The CP was incubated overnight at 5 molar excess over 10 nmoles ofG-actin in 400 μl of 5 mM Tris-HCl, pH 8.0, 1 mMATP 1 mM Ca Cl₂ and 10mM β-mercaptoethanol. The isopeptide linkage between the Lys-9 of CP andGln-41 of actin was formed by catalysis of 0.25 Units of guinea pigliver transglutaminase.

After overnight incubation at 4° C., the mixture was denaturated byboiling for 5 min and further digested with endoproteinase Lys C in thefollowing buffer: 25 mM Tris-HCl pH 8.5 1 mM EDTA with anenzyme/substrate ratio 1/50 by weight. The digestion was carried outduring 5 h at 37° C. and stopped by adding trifluoro acetic acid (TFA)to a final concentration of 0.2%. The peptide mixture was centrifugedand loaded (100 μl, corresponding to 84 μg (2 nmol) of actin) on a C-18reversed-phase column (4,6 mm×250 mm). Peptides were eluted with alinear gradient of acetonitrile (1.4% increase per minute) in 0.1% TFA(for details see FIG. 1A) and recorded by UV-absorption at 214 nm. Thepeptide elution profile of the endo Lys C digest on the actin-peptideconjugate is shown in FIG. 1A. Peptides were collected in 5 min. or 5 mlfractions and dried by vacuum centrifugation (Savant Instrument). Eachfraction was redissolved in 400 μl 40 mM Tris-HCl pH7.3, 50 mM NaCl andtreated with 0.12 Units of factor Xa (Promega). After digestion for 3 hat room temperature, TFA was added (final concentration 0.5%) and loadedon the same RP-chromatographic system. Of all the fractions analysed,only fraction 6 showed a shifting (shadow peak) peptide (FIG. 1B).

Electrospray-ionization mass spectrometry carried out with a Q-TOFMicromass instrument, confirmed the mass of the cross linked peptide(FIG. 2): Mm abs.: 3883.7 (Mm calc.: 3883.9), corresponding with the

dipeptide.

Edman-degradation further confirmed the sequence of the two cross-linkedchains: Cycle 1: Ala; Cycle 2: Gly+Asp; Cycle 3: Phe+Ser; Cycle 4: Ala;Cycle 5: Gly+Ser; Cycle 6: Asp; Cycle 7: Asp; Cycle 8: Ala; Cycle 9:Pro; with the ADSXS sequence from the CP and AGFAGDDAP-sequence derivedfrom 19-27 actin sequence.

This experiment showed the possibilities of the procedure and alsodemonstrated that the shift induced by the release of the NH₂-terminalpart of CP was sufficiently large to be useful in this invention.

1.6 Identification of the Target Site of a Compound on a SpecificProtein Present in a Highly Complex Mixture Such as a Cell Lysate.

Jurkat cells were lysed by incubation with 0.7% CHAPS, 0.5 mM EDTA, 100mM NaCl, 50 mM Hcpcs, pH7.5 and a protease inhibitor mix. This extractcontained 2 mg of total protein/ml.

Five hundred μl were desalted on a MAP5 disposable column equilibratedwith 25 mM Tris-HCl pH 8.5, 1 mM EDTA. To one ml of the desalted proteinmixture (1 mg), we added 50 μl of acetonitrile and 1.5 μg of endo Lys C.The digest was carried out for 5 h at 37° C.

Five hundred μl of this digest was mixed with 30 μl of the actin-CPendolysine C digest generated in the previous experiment (example 1.5)and 200 μl of 1% TFA in water was added. This mixture was centrifugedand loaded on a 4.6 mm×250 mm. RP-column (Vydac Separations Group).Peptides were eluted exactly as described in FIG. 1A. After 10 min.,fractions of 2 min. (2 ml volume) were collected during an additional 50min. In order to reduce the number of secondary runs, we pooled thefractions as indicated in Table 1.

Each of the combined fractions (A-E) according to Table 1 was vacuumdried and digested with factor Xa. This specific cleavage was carriedout in 2.5 ml of buffer containing 40 mM Tris-HCl pH7.3, 60 mM NaCl and0.12 Units of factor Xa protease. After 2 h, 100 μl of 1% TFA was addedand the mixture loaded on the same chromatographic system as in FIG. 1A.

Peptide elution was as in FIG. 1A. The peptide elution profile of pooledfraction D, containing the primary fractions 4-9-14-19-24 is shown inFIG. 3A. We observe peaks emerging from the intervals 9 and 14. Peak 9*eluting in front of interval 9 could not be identified as a peptide.Peak 9**, eluting on the tailing side of interval 9 is derived from theexcess of CP which did not react with actin. It is the NH₂-terminal partof CP with sequence Ac-Phe-Ile-Glu-Glu-Arg. This was confirmed by massspectrometry.

From interval 14 there is a new peak emerging in front of the bulk ofunmodified peptides (shown in black). This peak was identified as thecross-linked peptide

by mass spectrometry and Edman degradation (see also FIG. 2). No otherfractions showed peptides that shifted during the secondary run.

This experiment demonstrates that it is possible to specifically selectregions, segments or short sequences from proteins which are covalentlytargeted to compounds that interact with proteins via said regions,segments or short sequences. TABLE 1 Twenty-five fractions that werecollected in the first chromatographic separation, were collected infive pools (A-E) each containing the following combinations of primaryfractions: Pooled fraction N° Fraction numbers of primary run A 1 - 6 -11 - 16 - 21 B 2 - 7 - 12 - 17 - 22 C 3 - 8 - 13 - 18 - 23 D 4 - 9 -14 - 19 - 24 E 5 - 10 - 15 - 20 - 252. Differential Labeling of the Compound-Protein Complexes

2.1, the penicilloyl-moiety could carry one deuterium, more preferablytwo deuterium, more preferably three deuterium, more preferably fourdeuterium, preferably more than four deuterium atoms, replacing thecorresponding H-atoms in the “light” compound. It should be made clearhere that while it seems better to generate large mass differencesbetween the “light” and “heavy” species, for more accurate relativequantification, it is also clear that conversely co-elution orco-migration of the “light” and “heavy” forms of the tagged peptide inthe used chromatographic system is less probable with increasing massdifference. Thus the final used mass difference to discriminate between“light” and “heavy” compounds, should be a balance between the largestmass difference for accurate relative quantification and differencesstill giving rise to identical or very similar chromatographicproperties.

2.2, one or more of the amino acids specifying the caspase-1 inhibitoryactivity could be substituted by an equivalent deuterated amino acid.For instance, the Valine residue could be substituted for d₇-Valine ord₈-Valine. Alternatively, the Alanine residue could be substituted byd₃-Alanine. Thus in sample one, the “light” compound will be linked toits biological targets), while in sample two, the same compound, but nowwith one or more amino acid(s) substituted by their deuterated homologswill be linked to the biological targets. The peptide mixtures,including the compound-target peptides of samples one and two are mixed.The tagged peptides co-elute and are co-transferred to the massspectrometer in which they segregate due to their mass differences. Theion intensities, corresponding to both masses are used to calculate theratios of both tagged peptides, thus of both protein levels or/andactivity levels.

2.3, differential labeling will be most conveniently at the phenylgroup, present in the sulfenylbenzoyl tag of FSBA. This group canharbour four deuterium atoms. Similar to what is described in theprevious examples, protein mixture 1 will be labeled with the H⁴-FSBAreagent (light reagent) while protein mixture 2 will be labeled with FS4dBA (heavy reagent). After sorting, both of the light and heavy taggedpeptides can be compared based on the relative intensities of theirrespective ions after separation by mass spectrometry.

1. A method to isolate at least one target molecule of a compoundcomprising a functional group that can be specifically altered, saidmethod comprises the following steps: (a) adding said compound to acomplex mixture of molecules wherein said compound stably interacts withat least one molecule forming a compound-target complex, (b) separatingthe resulting complex mixture of molecules and compound-target complexesinto fractions via chromatography, (c) chemically, or enzymatically, orchemically and enzymatically altering said compound present on at leastone compound-target complex in each fraction, and (d) isolating at leastone target molecule that interacts with said compound viachromatography, wherein the chromatography of steps (b) and (d) isperformed with the same type of chromatography.
 2. The method of claim1, wherein the chromatographic conditions of steps (b) and (d) are thesame or substantially similar.
 3. A method according to claims 1 whereinsaid complex mixture of molecules is a complex mixture of proteins.
 4. Amethod according to claim 3 further comprising the cleavage of saidcomplex protein mixture into a protein peptide mixture before performingstep (b).
 5. A method according to claims 1 wherein said complex mixtureof molecules is a protein peptide mixture.
 6. The method of claim 1,further comprising the step of identifying the targets.
 7. The method ofclaim 6, wherein said target molecules are proteins or peptides andwherein said identifying step is performed by a method selected from thegroup consisting of: a tandem mass spectrometric method, Post-SourceDecay analysis, measurement of the mass of the peptides, in combinationwith database searching.
 8. The method of claim 7, wherein saididentifying step based on the mass measuring of the target peptides isfurther based on one or more of the following: (a) the determination ofthe number of free amino groups in the target peptides; (c) theknowledge about the cleavage specificity of the protease used togenerate the protein peptide mixture; and (d) the grand average of thehydropathicity of the target peptides.
 9. A method to determine therelative amount of the level and/or activity of at least one targetprotein in more than one sample comprising proteins, comprising thesteps of: (a) the addition of a compound comprising a first isotope to afirst sample comprising peptides wherein said compound stably interactswith at least one peptide forming a compound-peptide complex; (b) theaddition of a compound comprising a second isotope to a second samplecomprising peptides wherein said compound stably interacts with at leastone peptide forming a compound-peptide complex; (c) combining theprotein peptide mixture of the first sample with the protein peptidemixture of the second sample; (d) separating the combined proteinpeptide mixtures into fractions of peptides via chromatography; (e)chemically, or enzymatically, or chemically and enzymatically, alteringsaid compound present on at least one compound-peptide complex in eachfraction; (f) isolating the altered compound-peptide complexes out ofeach fraction via chromatography, wherein the chromatography isperformed with the same type of chromatography as in step (d); (g)performing mass spectrometric analysis of the isolated alteredcompound-peptide complexes; (h) calculating the relative amounts of thealtered compound-peptide complexes in each sample by comparing the peakheights of the identical but differentially, isotopically labelledaltered compound-peptide complexes, and (i) determining the identity ofsaid peptides in the altered compound-peptide complexes and theircorresponding proteins.
 10. The method according to claim 9 wherein thechromatographic conditions of steps (d) and (f) are the same orsubstantially similar.
 11. The method of claim 9 or 10 wherein thedetermining of the identity of the altered compound-peptide complexes isperformed by a method selected from the group consisting of: the tandemmass spectrometric method, Post-Source Decay analysis, measurement ofthe mass of the peptides, in combination with database searching. 12.The method of claim 11, wherein the determining of the identity of thealtered compound-peptide complexes is further based on one or more ofthe following: (a) the determination of the number of free amino groupsin the peptides; (c) the knowledge about the cleavage specificity of theprotease used to generate the protein peptide mixture; and (d) the grandaverage of the hydropathicity of the peptides.